Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

• the present invention relates to polynucleotide probes and a method for their preparation.
• the polynucleotide probes comprise a polynucleotide moiety, a hydrazide compound, and a reporting moiety.
• the reporting moiety is attached to the hydrazide compound which in turn is attached to the 4-position of a deaminated cytosine base.
• a number of diagnostic methods are available for the detection of pathogens or infectious agents.
• the traditional method comprises cultivating the pathogen until it could be identified, for example, by morphology and/or color.
• This method has drawbacks in that not all pathogens can be cultivated, and in that the method is time consuming, laborious, relatively non-specific, and relatively insensitive.
• a most promising technique for identifying pathogens is that of nucleic acid hybridization. Besides being specific, sensitive, rapid, simple, and versatile, the method allows the identification of pathogens in aged or in highly contaminated samples. This method is also not limited to the detection of pathogens. It can be used to detect the presence of any target polynucleotide, for example, an oncogene.
• a chemical method for the preparation of polynucleotide probes thus has many advantages.
• Initial attempts to chemically label polynucleotides involved the introduction of a label onto the terminal sugar of a polynucleotide.
• a fluorescent label was attached to the 3 ⁇ terminal end of RNA (Reines and Cantor ((1974)), Nucleic Acids Res., 1 :767-786, and Wangyi et al. ((1980)), Scientia Sinica 23 :1296-1308) and to the 5 ⁇ terminal end of DNA (Smith et al. ((1985)) Nucleic Acids Res., 13 :2399-2412).
• a biotin label was also attached to the 5 ⁇ terminus of oligodeoxynucleotides.
• Another paper described the attachment of a fluorescent moiety to a tRNA.
• the purpose was to study the effect of modifications of bases on the amino acid acceptor activity of tRNA's and to determine which cytosine bases, if any, are involved in recognition by the synthetase enzyme.
• the method involved the displacement of the 4- amino group of cytosine with carbonyl hydrazide followed by attachment of fluorescein to the hydrazide.
• the report did not involve a study on the effect of the attached fluorescent moiety on the base-pairing ability of the cytosine to guanine or on the use of such a polynucleotide as a probe.
• bridging moieties of potential diagnostic importance, such as biotin were not added.
• N-acetoxy-N-2- acetylaminofluorene is a carcinogen
• a probe comprising this compound is hazardous to prepare and use besides having safety and disposal problems. See Landegent et al. (1981), Exp. Cell Res., 153 :61-72 and Tchen et al. (1984), Proc. Natl. Acad. Sci. USA, 81 :3466-3470.
• the crosslinking of DNA with haloalkylhydrazides has been carried out.
• the crosslinking was between cytosine and gaunosine.
• the object was to develop a general method for blocking specific genetic sequences to study in vitro the mechanisms of replication, transcription, and repair. No reporter molecule was attached to the hydrazide. See "Sequence-specific Crosslinking Agents for Nucleic Acids, use of 6-Bromo-5,5-­dimethoxyhexanohydrazide for Crosslinking Cytidine to Guanosine and Crosslinking RNA to Complementary Sequences of DNA", by J. Summerton and P.A. Bartlett, J. Mol. Biol. (1978) 122 :145-162.
• a method for preparing a probe comprising a reporting moiety attached to the 4-position of cystosine is briefly alluded to in European Patent Publication No. 0,097,373, published in January 4, 1984 by Dean Engelhardt et. al. However, no specific details are given other than that the 4-amino group of cytosine can be alkylated by a reporting moiety.
• a synthetic method for the preparation of a polynucleotide comprising a hydrazide compound.
• the hydrazide is attached to the 4-position of a former cytosine moiety to form a cytosine analog by displacing the 4-amino group.
• This method permits the synthesis of a polynucleotide probe, perferably, a polydeoxynucleotide probe, by attaching a reporting moiety to the hydrazide compound by means of a second functional group present in the hydrazide compound in which instance the hydrazide compound serves as a linker arm for attaching the reporting moiety to the polynucleotide.
• the hydrazide compound can be attached to the 4-position of a cytosine base of a polynucleotide or to the 4-position of a cytidine triphosphate which is then incorporated into a nascent polynucleotide.
• a reporting moiety or bridging moiety can be a joined to a hydrazide linker arm that is attached to the 4-position of a cytidine triphosphate and the modified cytidine triphosphate can then be incorporated into a nascent polynucleotide chain.
• the polynucleotide probe can be prepared by either (a) reacting the hydrazide compound with a polynucleotide and attaching a reporting moiety to the hydrazide compound, or (b) attaching the reporting moiety to the hydrazide compound (or converting a functionality in the reporting moiety to a hydrazide functionality) and reacting the hydrazide compound with a polynucleotide.
• polynucleotide probes preferably, deoxypolynucleotide probes
• a hydrazide compound with a polynucleotide, preferably a polydeoxynucleotide, to displace an amino group of at least one cytosine base from said polynucleotide and attaching a reporting moiety to said hydrazide compound.
• This invention relates to a method for the preparation of an entity comprising a polynucleotide and a hydrazide compound.
• the entity is prepared by displacing the 4-amino group of a cytosine moiety with the hydrazide functionality in the absence of bisulfite.
• This invention also relates to a method for chemically preparating a polynucleotide probe.
• the polynucleotide probe generally comprises a polynucleotide moiety and a reporting moiety which is attached to the polynucleotide moiety.
• the polynucleotide moiety of the probe has the ability to base-pair, i.e., hydridize to a complementary polynucleotide sequence of interest or target polynucleotide.
• the reporting moiety of the probe has or produces the means by which the presence of the target polynucleotide can be verified.
• the means can be, for example, fluorescence, phosphorescence, radioactivity, chromogen, electron density, or chemiluminescence.
• the polynucleotide probe can be prepared in several ways.
• One way which is preferred, is to react a hydrazide compound in the absence of bisulfite with a polynucleotide to displace a 4-amino group of at least one cytosine base.
• a reporting moiety is then directly attached to the hydrazide compound by means of a functional group other than the hydrazide present in the hydrazide compound.
• a reporting moiety is first attached to the hydrazide compound, and the hydrazide compound reacted with a polynucleotide to displace an amino group of at least one cytosine base.
• Other variations comprise attaching a reporting moiety indirectly to the hydrazide compound by means of a bridging moiety.
• the hydrazide compound serves as a linker arm for linking the polynucleotide to the reporting moiety.
• the polynucleotide containing the desired base sequence can be prepared enzymatically or synthetically.
• Enzymatic preparation involves the use of an appropriate template, a polymerase enzyme ans a mixture of the triphosphate precursors including one whose base portion is modified.
• One suitable method uses the M13 vector. These methods are well known to one skilled in the art and will not be discussed here.
• Synthetic methods involve attaching a linker arm to a predetermined unmodified polynucleotide. Such methods can provide a simple and easy method for large scale production of DNA probes. Inexpensive, readily available chemicals can be employed and theoretically, no special equipment is needed.
• the number of linker arms attached to the polynucleotide can be controlled by varying the amounts of each reactant and the reaction conditions.
• Synhthetic methods also have the unique advantage in that the synthesis of the polynucleotide can be automated by instrumentation. It is conceivable that automation will permit the incorporation of a chemically synthesized nucleotide precursor containing a linker arm directly into a specific base sequence.
• the polynucleotide need not be prepared synthetically. It can be prepared enzymatically with polymerase enzyme by methods well known to those skilled in the art.
• the most widely used chemical synthesis involves the phosphoramidite method because of its inherently high coupling efficiency and the stability of the starting materials.
• the starting material is the solid support derivatized with a 3 ⁇ -hydroxyl terminal nucleoside.
• the nucleoside is attached to the silica to the 3 ⁇ -OH.
• the 5 ⁇ -hydroxyl is blocked with a dimethoxytrityl group.
• the synthesis is performed with the growing polynucleotide chain attached to a solid support so that excess reagents whcih are in the liquid phase can be removed by filtration, eliminating the need for purification steps between base additions.
• the first steps of the synthesis cycle comprise:treating of the derivatized solid support with acid to remove the trityl group and generate a free 5-OH; activating a predetermined nucleoside by adding its phosphoramidite derivative and a weak acid, tetrazole, to the reaction chamber, and reacting the freed 5 ⁇ -OH with the activated nucleoside.
• the addition reaction is generally complete in less than two minutes at room temperature.
• the next steps comprise: capping all sequences which did not undergo addition, and converting all phosphite internucleotide linkages to the more stable phosphates. This reaction is 100% complete in less than 30 seconds.
• the dimethoxytrityl group is removed and the cycle is repeated until the chain elongation is complete.
• the oligo­nucleotide is still bound to the solid support and has protecting groups on the phosphates and on the exocyclic amino groups of the bases A, G, and C.
• the methyl groups on the phosphates are removed and the chains are cleaved from the support. After the solution containing the DNA is removed from the instrument, the protecting groups on the exocyclic amines of the bases are cleaved.
• the degree of subsequent purification required depends on the application. With the 98-100% stepwise yields routinely obtained with the synthesizer, it is often not necessary to purify probe oligonucleotide containing from about 15 to about 25 bases. Other applications using longer oligonucleotides may require purification by electrophoresis or high pressure liquid chromatography (HPLC).
• HPLC high pressure liquid chromatography
• Polynucleotide synthesis using the phosphoramidite method provides a number of advantages. Firstly, the diisopropyl phosphoramidites are stable for a prolonged period and are nonhygroscopic. Secondly, coupling reactions using phosphoramidites can be carried out in acetonitrile (rather than in pyridine) which favors the keto form, thus protecting the bases from side-chain addition.
• the polynucleotide should comply with at least about 12 to 15 bases. This is to impart specificity to the base sequence and make it selective as a probe. However, according to one manufacturer (Applied Biosystems), one can prepare polynucleotides up to about 200 bases.
• the hydrazide compound is attached to a polynucleotide by displacing the 4-amino group of a cytosine moiety.
• the hydrazide compound can serve as a linker arm for attaching a polynucleotide moiety.
• the hydrazide compound when serving as a linker arm can contain as little as 6 atoms or can contain over 1000 atoms, preferably from about 6 atoms to about 100 atoms.
• the linker arm can be attached to the cytosine bases of either single-stranded polynucleotides or double-stranded polynucleotides that have been partially denatured.
• the linker arm comprises a first hydrazide functionality and a second functionality other than a primary amino group that is attached to the carbon alpha to the carbonyl of the hydrazide, provided that the second functionality does not substantially react with the first hydrazide functionality of either the same or another molecule.
• the functionality can be a nucleophile, an electrophile, or a leaving group.
• Examples of functionalities include but are not limited to carboxylic esters, carboxylic acids thioesters, imides, ketones, aldehydes, epoxides, halides, n-hydroxysuccinimide esters, isocyanates, isothiocyanates, imidates, anhydrides, hydrazines, alkoxides, alkylamines, mercaptans, carboxylates, sulfonates, hydrazides, haloketones, diazonium salts, acylazides, nitrophenyl esters, sulfonyl chlorides and maleic imides.
• Preferred second functionalities include hydrazines, amines, alkylamines, mercaptans, carboxylates, and carboxylic esters. Particularly preferred second functionalities include hydrazines, amines, and alkylamines.
• the hydrazide compound can comprise, for example, a carbon-carbon double bond, carbon-nitrogen single bond, carbon-nitrogen double bond, carbon-carbon triple bond, carbon-oxygen single bond, carbon-sulfur single bond, or carbon-silicon single bond.
• Preferred hydrazides compounds are those that are soluble in aqueous solutions.
• a particularly preferred hydrazide is carbonyl hydrazide.
• Other preferred hydrazides are amino acid hydrazides wherein the amino group is not or to the carbonyl group, and monohydrazides of dicarboxylic acids. It should be noted that oxlayl hydrazide is less preferred because of its poor solubility in aqueous media at the optimum pH required for reaction, while malonic and tartaric di­hydrazides are less preferred because they decompose in aqueous solution.
• the linker arm for attaching a reporting moiety to a polynucleotide is a hydrazide compound.
• the 4-amino group of cytosine is displaced by the hydrazide compound in a displacement reaction.
• the conditions for the displacement reaction will vary with the particular hydrazide. Factors that affect the reaction are solubility of the hydrazide in aqueous solutions, reactivity of the hydrazide, and stability of the hydrazide under the reaction conditions. Hydrazides that are water soluble are preferred because the displacement reaction can be carried out in aqueous solutions. However, the reaction can also be carried out in a mixed solvent system.
• the pH range for the reaction can vary from about 3.0 to about 8.0, although from about 3.5 to about 6.0 is preferred.
• the temperature can vary from about 20°C to about 90°C, although from about 35°C to about 50°C is preferred.
• any buffer can also be present.
• the presence of bisulfite is not required - in fact, it is not desired, because it is a mutagen.
• the stoichiometry of the reactants can vary. Generally, an excess of the hydrazide compound will be employed as compared to the number of cytosine bases. However, an expensive hydrazide compound may dictate the use of a stoichiometric ratio or even for an excess of base.
• the reporting moiety provides the means for detecting the presence of the target polynucleotide.
• the reporting moiety can be attached directly to the linker arm (hydrazide compound), or indirectly by means of one or more bridging moieties.
• the reporting moiety or bridging moiety can be attached to the linker arm or to a bridging moiety covalently or non-covalently.
• the reporting moiety can be attached to the linker arm following reaction of the linker arm with the polynucleotide or prior to reaction of the linker arm with the polynucleotide.
• the reporting moiety can be attached directly and covalently to the linker arm by anyone of a number of functionalities present in the reporting moiety.
• functionalities include, but are not limited to, isothiocyanates, carbodiimides, sulfonyl chlorides, and n-hydroxysuccinimide esters.
• the functionality should be capable of reacting with the second functional group of the linker arm, i.e., the one other than the hydrazide functionality.
• the second functionality of the linker arm which is attached to a cytosine of a polynucleotide is a an amino group
• the reporting moiety comprises an isothiocyanate functionality.
• the amino group reacts with the isothiocyanate to attach directly and covalently the linker arm to the reporting moiety.
• a number of reporting moieties are commercially available which already have the required funtional group by means of which the reporting moiety can be become attached to the linker arm. See, for example, the 1984 Catalog by Enzo Biochem. Inc., 325 Hudson Street, New York, New York 10013.
• the reporting moiety can also be attached to the linker arm indirectly and non-covalently. This can be, for example, where alkaline phosphatase, the reporting moiety, is covalently attached to an avidin molecule. Biotin, a bridging moiety, is covalently attached to a linker arm which is attached to a cystosine of a polynucleotide. The formation of a complex between biotin and avidin results in the reporting moiety becoming indirectly and non-covalently attached to the linker arm.
• reporting moieties which are enzymes include, but are not limited to, hydrolases, esterases, phosphatases, peroxidases, catalases, glycosidases, oxidoreductases, proteases, lipases, and nucleases. Particularly prefered are the phosphatases, peroxidases, and oxidoreductases.
• fluorescent moieties include, but are not limited to, rhodamine B and fluorescein.
• reporting moieties there are no limitations as to how many reporting moieties can be attached to the polynucleotide. The greater the number of reporting moieties, the greater is the sensitivity of the polynucleotide probe. However, in order to minimize steric hindrance, it is believed that they are not be more than one reporting moiety per four sequential nucleotides.
• Polynucleotide hybridization is based on complementary base-pairing.
• target polynucleotides are mixed either in solution or on a support with single-stranded polynucleotide probes, complementary base sequences pair to form double-stranded hybrid molecules.
• Detection of target polynucleotides by their hydridization to polynucleotide probes can be carried out, for example, by isolating the double-stranded polynucleotides from a sample and fixing them onto a support, for example, nitrocellulose, glass slide, plastic, paper or other synthetic polymer.
• the fixed polynucleotides are mixed with a solution containing the polynucleotide probe, and the support is heated to about 80-90°C to denature the polynucleotide double-strands. (The double-strands can also be denatured by means of alkali).
• the system which now contains the denatured target polynucleotide and the polynucleotide probe is allowed to cool to an appropriate temperature, to allow hybridization to take place. After sufficient time has elapsed for hybridization to be complete, which can be for ten minutes to several hours, the fixed target polynucleotide is washed to remove all unbound polynucleotide probes.
• the reporting moiety of the polynucleotide probe is now detected, either directly, for example, by means of radioactivity or fluorescence, or indirectly, for example, by means of a chromogen formed through an enzymatic reaction.
• a chromogen formed through an enzymatic reaction See M. Grunstein and J. Wallas, METHODS IN ENZYMOLOGY, volume 68, R.Wu (Ed) (1979) pp. 379-469; A.R., Dunn, and J. Sambrook, METHODS IN ENZYMOLOGY, volume 65; part 1, (1980) pp. 468-478; Modified Nucleotides And Methods Of Preparing and Using The Same By D.C. Ward, A.A. Waldrop, and P.R.
• a second way is to detect the target polynucleotide in - situ .
• the tissue specimen comprising the target polynucleotide is fixed to a glass slide.
• the specimen can be fixed, for example, either in acetone or in CARNOY's B fixative (10% acetic acid, 30% chloroform, 60% methanol).
• the fixative is allowed to remain in contact with the specimen for about 5 minutes. Excess fixative is tapped off the slide and the slide is allowed to air dry.
• a solution containing a polynucleotide probe with a fluorescent molecule as its reporting moiety is added to the specimen on the support and the specimen is covered with a coverslip.
• the covered slide is heated to about 90-95°C for approximately 0.5-2 minutes to denature all the double-stranded polynucloetides.
• the slide is then removed from the heating block and hybridization is allowed to proceed at room temperature for about 10 minutes.
• the specimen is first washed with stringency solutions to remove all nonhybridized polynucleotide probes and then washed with buffer to remove the stringency solution.
• the slide is finally examined under a fluorescent microscope. The presence of fluorescence in the specimen indicates the presence of the target polynucleotide.
• the reporting moiety can be an enzyme.
• the enzyme cannot be present during the heating step because it would become denatured by the heat.
• the enzyme can be added however following the stringency wash.
• An example of such a method is where biotin, a bridging moiety, is attached to a linker arm which is attached to cytosine, and the enzyme reporting moiety is attached to avidin.
• the polynucleotide probe comprising the biotin is allowed to mix with the specimen is added to the fixed specimen following the stringency wash.
• the presence of the target polynucleotide will result in the formation of a polynucleotide hybrid wherein one of the polynucleotides comprises biotin.
• the addition of the avidin to which the enzyme is bound to the specimen results in the avidin complexing to the biotin.
• the addition of a substrate to the enzyme and the appearance of a color verifies the presence of the target polynucleotide.
• Carbohydrazide (purchased from Aldrich Chemical Comp.) solution was freshly prepared by dissolving 0.36 g of carbohydrazide in 1.85 ml of distilled water and slowly adding 0.15 ml of concentrated HCl. The final concentration of carbohydrazide was 2M and the final pH of the solution was between 4.15 and 4.25.
• carbohydrazide modified DNA (CH DNA) in a mixture of 100 ul of 90 mM borate buffer (pH 8.5) containing 10 mM EDTA and 30 ul of DMSO was added 30 ul of biotin solution (40mg/ml in DMSO). The mixture was kept at room temperature overnight. The modified DNA was then precipitated by the addition of 320 ul of cold alcohol. After 20 hours at -20°C, the DNA was collected by centrifugation and then washed once with 300 ul of cold alcohol (70%). After lyophilization, the biotinylated DNA probe was stored in 150 ul of tris EDTA buffer (10 mM tris HCl (pH 7.5) mM EDTA).
• 125 ug of carbohydrazide modified M13 DNA was dissolved in 200 ul of borate buffer, and 100 ug of fluorescein isothiocyanate (FITC) solution (10mg/ml DMF) was added. The reaction mixture was incubated at room temperature for 20 hours. The reaction mixture was purified by passage through a G-50 column (10 cm ⁇ 1 cm), using 50 mM triethylammonium bicarbonate (pH 7.5) as eluting buffer. The fluorescein labelled DNA was obtained by lyophilization of the solvent. The method for the measurement of the number of fluorescein molecules on the DNA was adapted from that of Reines, S.A. and Schulman, L.H., Methods Enzymol. (1959) 59 :146. This measurement indicated that one fluorescein molecule was incorporated per 10 to 15 bases of DNA.
• FITC fluorescein isothiocyanate
• the labeling procedure was adapted from that of Titus, J.A., Hangland, R., Sharrow, S.O., and Sega., D.M., J. Immunol. Methods (1982) 50 :193.
• One hundred twenty five ug of carbohydrazide-modified-M13 DNA was dissoved in 250 ul of borate buffer (90 mM, pH 8.5) containing 10 mM of ethylenediaminetetraacetic acid (Na+, pH 7.5.).
• Ten mg of Texas Red-celite mixture (w/w, 1:9) was suspended in the DNA solution. The reaction mixture was stirred at 4°C in the dark overnight (about 16 hours).
• Texas-Red labeled DNA was purified by elution with 10 mM of triethylammonium bicarbonate, pH 7.5, through a G-50 column (0.9 ⁇ 10 cm). The eluate was removed by repeated lyophilization. The DNA thus obtained was assayed by measurement of the ratio of the absorption at 590 nm to 260 nm. This measurement indicated that one fluorophore was incorporated per 10 to 15 bases of DNA.
• the biotinylated M13 probe (0.25 ug/ml), prepared as described above, was added and hybridization was carried out at 37°C for 16 hours in hybridization buffer (30% deionized formamide in 4 X SSPE, 5% dextran sulfate, 1% triton X-100 and 1mg/ml of sonicated, heat denatured salmon sperm DNA).
• hybridization buffer (30% deionized formamide in 4 X SSPE, 5% dextran sulfate, 1% triton X-100 and 1mg/ml of sonicated, heat denatured salmon sperm DNA.
• the plate was washed twice with 2 X SSC, 0.1% triton X-100 at 65°C.
• the plate was incubated at 37°C for 30 minutes with blocking solution (20ug/well, PBS (Phospate Buffered Saline), 2% Bovine Serum Albumin, 0.1% of Triton X-100 and 5mM EDTA). After the blocking solution was removed, the plate was incubated at 37°C for 30 minutes with Horseradish-streptavidin complex (diluted 1:5,000 in PBS, 1% Bovine Serum Albumin, 0.1% Triton X-100 and 5mM EDTA; 50ug/well).
• blocking solution 20ug/well, PBS (Phospate Buffered Saline), 2% Bovine Serum Albumin, 0.1% of Triton X-100 and 5mM EDTA.
• a solution of o-phenylenediamine which was used as substrate for horseradish peroxidase was prepared by dissolving 4mg of o-phenylenediamine and 25 ul of H2O2 (10%) in 20 ml of phosphate-citrate buffer (pH 6). The plate was incubated with this o-phenylenediamine solution for 5-10 minutes at room temperature and the reaction was stopped by adding 4N sulfuric acid solution. The detection of the target DNA was measured by reading the visible light absorbance at 490nm.
• the sensitivity of the biotinylated M13 DNA probes was compared with that of biotin nick-translated M13 probe, prepared by the procedure of Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Proc. Natl. Acad. Sci. USA 78 :6633.
• the chemically biotinylated M13 DNA probe (prepared as described above) was at least two times more sensitive than the nick-translated M13 DNA probe (with 24% of the thymidine nucleotides substituted with Bio-11-dUTP) in the detection of target M13 RF DNA.
• the fact that a sensitive, specific probe could be produced by this labeling method indicates that hydrogen bonding of the modified cytosine bases to the complementary guanine residues must be taking place.
• M13 RF DNA (24ug/80ul in tris-HCl (10 mM pH 7.5)-EDTA (1 mM) was heat denatured by boiling it at 100°C for 5 minutes and then cooling it to 0° in ice water. The DNA was then dissolved in 400 ul of 2M carbohydrazide solution. The mixture was incubated at 37°C for 6 hours and the excess reagent was removed by repeated alcohol precipitation as described above. Twenty ug of carbohydrazide modified DNA was obtained (83%).
• Carbohydrazide-modified double-stranded M13 (RF) DNA (20ug in 50ul of distilled water) was heat denatured and then added to 0.15 ml of a solution containing borate buffer and DMSO (2:1). To the above solution was added 50ul of biotin-aminocaproic acid N-hydroxy succinimide ester (ENZOTIN) solution (40 mg/ml in DMSO). The mixture was incubated for 15 hours at 37°C and the biotinylated M13 RF DNA was isolated by the standard alcohol precipitation procedure. Ten ug of biotinylated DNA were obtained (50% yield).
• Filters were then pre-hybridized at 65°C for at least 2 hours in pre-hybridization buffer (5 X SSC, 0.1% SDS, 1.0 mg/ml each of bovine serum albumin, ficoll 400 and polyvinylpyrrolidone, MW, 40,000, and 1 mg/ml of sonicated, heat denatured [boiled for 10 minutes and quickly chilled in ice-water] salmon sperm DNA).
• Hybridization was carried out in a shaken water bath at 65°C for 20 hours in the pre-hybridization buffer containing 50 ng/ml of heat denatured biotin-labelled M13 RF probe. Filters were washed while shaken in 2 X SSC, 0.1% SDS (2 x 15 minutes, at 65°C), and then washed twice at room temperature with 2 X SSC solutions.
• the colorimetric detection method was adapted from that of Leary, J.J., Brigati, D.J. and Ward, D.C. (1983) Proc. Natl. Acad. Sci. 80 :4045.
• the filters were incubated at 37°C for 30 minutes with horseradish peroxidase-streptavidin complex in the buffer containing phosphate buffered saline (0.13 M of sodium chloride, 0.007 M dibasic sodium phosphate and 0.003 M monobasic sodium phosphate and 1% of bovine serum albumin).
• the filter After washing the filters three times with high salt buffer (0.5 M sodium chloride, 10mM phosphate buffer [pH 6.5], 0.1% bovine serum albumin, 0.005% Tween-20, the filter was developed with diaminobenzidine tetrahydrochloride in 10 mM Tris buffer (pH 7.5). The reaction was terminated by washing the filters with tap water and blotting them dry.
• the sensitivity of detection of target double-stranded M13 RF DNA was 400 pg. Under the same conditions, the sensitivity of detection using a biotin nick-translated M13 probe was 80 pg of target M13 RF DNA.
• the reaction mixture was diluted to 400 ml with water and then treated with 10ml of 0.5N lanthanum nitrate. The mixture was kept in a cold room for 3 hours and the white precipitate was collected by centrifugation. The supernatant was decanted off, and the white precipitate was suspended in 250 ml of water and 25 g of Dowex 50- X 8 (Na+) was added. The mixture was stirred continuously until a clear solution resulted. By paper chromatography, as described earlier, this solution was shown to contain a single compound which was positive to the hydrazide test (spraying with reagent containing 1 mg picryl sulfonic acid in 1 ml of 0.1 M Na borate buffer generated an orange red color.
• the cabohydrazide modified CMP was obtained as white powder after lyophilization.
• a sample was further purified by passage through an anion exchange column (DE52 column, eluted with a linear gradient from 0.01 M to 0.3 M triethyl-ammonium bicarbonate, pH 7.5).
• the desired product was eluted from the column at about 0.08 M triethylammonium bicarbonate and lyophilized to give a white powder.

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